1. Field of the Invention
The present invention relates generally to the study of herpesviruses. More specifically, the present invention relates to clinical assays for the detection and typing of human herpesviruses.
2. Description of the Related Art
There are more than 100 known herpesviruses in the family of Herpesviridae. Of these, eight are known to infect humans. The eight human herpesviruses are herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpesvirus 6 (HHV-6), herpesvirus 7 (HHV-7), and herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma associated herpesvirus (KSHV).
Based on the length of viral replication cycle and host tissue range, the herpesviruses are classified into 3 sub-families (alpha-, beta-, & gamma-herpesviruses, Table 1). Following primary infection, all herpesviruses establish latent persistent infections within tissues characteristic for each virus. For example, the alpha-herpesviruses HSV1, HSV2 and VZV are neurotropic, while EBV, CMV, HHV6, HHV7 and HHV8 are lymphotropic.
All herpesviruses share certain characteristics. All are composed of a core of double-stranded DNA encased within an icosahedral capsid and a phospholipid bilayer envelope. Human herpesvirus infections are very common and widely distributed. Serologic surveys indicate that >95% of adults worldwide have been infected by VZV, EBV, and HHV-6.
Despite a vigorous anti-viral immune response, herpesviruses persist in the host following primary infection. This asymptomatic latent period may be interrupted by periods of viral reactivation during which virus replicates and clinical symptoms may occur. Examples include recurrent cold sores (HSV-1), herpes zoster (shingles) in older adults arising from VZV acquired during childhood (chicken pox), CMV pneumonitis in immunocompromised organ transplant patients, and recurrent mononucleosis in patients with chronic (EBV) mononucleosis syndrome.
In many cases, the diagnosis of herpesvirus infection cannot be accurately made by clinical findings alone. Symptoms are often nonspecific, e.g. fever, malaise, lymphadenopathy, and rash. Patients can sometimes be infected with more than one herpesvirus (e.g. frequent association of HHV-8 and EBV in primary effusion lymphoma, HSV-1 and HSV-2 in orogenital ulcers). Whereas infections with the _-herpesviruses and CMV are usually amenable to acyclovir or gancyclovir anti-viral treatment, no clearly effective drug treatment is available for EBV, HHV-6, HHV-7, and HHV-8. Thus, identification of specific human herpesvirus infection is necessary before proper therapy can be selected.
The pathogenesis and clinical importance of the recently identified lymphotropic viruses HHV-6, HHV-7 and HHV-8 are not well understood. A better clinical understanding of these viruses requires the availability of appropriate diagnostic approaches for their detection and identification. All these factors, along with the worldwide impact of human herpesvirus infection, drive the need for a reliable multiplex clinical assay for the detection and identification of all eight human herpesviruses. Although a clinical assay need not differentiate EBV-1 from EBV-2 or HHV-6A from HHV-6B, it should be able to detect each variant and to distinguish all strains of EBV and HHV-6 from the other herpesviruses.
Current laboratory techniques for detection of herpesvirus infection include virus culture, viral serology, and viral DNA detection by PCR. Given the lack of optimal methods, viral culture is not generally available for detection of EBV, HHV-6, HHV-7, and HHV-8 infections. Culture detection of HSV, VZV, and CMV often suffers from poor sensitivity and very slow turn-around time. Serologic testing for HHV-6, HHV-7, and HHV-8 are not widely available, and due to their close antigenic similarity, distinction of HSV-1 from HSV-2 infection by serologic methods is often unreliable. In addition, in many cases it is difficult to serologically distinguish between acute infection and normal baseline seropositivity, thus necessitating the inconvenience of obtaining both acute and convalescent titers. Given the considerable limitations of culture and serology for herpesvirus detection, PCR detection methods have been developed. PCR offers the distinct advantages of rapid turn-around time, high sensitivity, and high specificity for the detection of herpesvirus infections.
Significant interest in the molecular evolution of herpesviruses led to sequencing of the genomes of many of the human and non-human herpesviruses. Although there are substantial differences between the genomes of different human herpesviruses, certain highly conserved regions, including the DNA polymerase gene, have been identified. This information has been utilized to develop multiplex PCR assays using consensus primers to conserved regions of the herpesvirus DNA polymerase gene.
Rozenberg and Lebon (1991) described a single step PCR assay using a consensus primer pair for HSV-1, HSV-2, EBV, and CMV, followed by typing of the amplicons by restriction fragment length polymorphism analysis (RFLP). However, the complexity of RFLP restricts its use to sophisticated laboratory environments. Moreover, the consensus primer set did not amplify VZV or HHV-6. Tenorio et al. (1993) revised this approach to include amplification of VZV, yet again typing the amplicons with RFLP. Aono et al. (1994) described a multiplex PCR using consensus primers for the detection of the three _-herpesviruses (HSV-1, HSV-2 and VZV) by virus-specific probe hybridization. Unlike restriction fragment length polymorphism, virus-specific probe hybridization is more compatible with clinical laboratory requirements of short cycle time and simplicity.
van Devanter et al. (1996) developed a set of degenerate consensus primers for PCR amplification of conserved regions of the DNA polymerase gene. The resulting nested consensus primer PCR method allowed for amplification and identification of most (14 of 15) of the animal herpesviruses and 6 of 8 human herpesviruses (HHV-1, HHV-2, VZV, EBV, CMV, HHV-6B). The method did not amplify human DNA polymerase, or yeast/mold DNA polymerase that are common contaminants of human samples. However, the methodology exhibited a wide variation in sensitivity across the human herpesviruses tested. The LOD (limit of detection) varied between 1 copy per 100 ng DNA for HSV-1 and HSV-2, and 100 copies for EBV and VZV. No data was presented on amplification of HHV-7 or HHV-8. van Devanter identified each virus by direct DNA sequencing of the amplified products obtained from an ethidium bromide stained agarose gel. This method of DNA sequence typing is a highly complex, laborious method not appropriate for use in a clinical diagnostic laboratory. Moreover, van Devanter did not demonstrate that the method could identify more than one herpesvirus in a single sample.
Ehlers et al. (1999) developed an enhanced version of the van Devanter method. Ehlers noted that the van Devanter method exhibited a wide variation in binding of the degenerate primers to different herpesviruses. Knoth et al. (1988) had previously shown that reducing primer degeneracy with deoxyinosine (dI) improved the performance in DNA amplification from related species. Ehlers thus substituted deoxyinosine (dI) at the 3- and 4-fold degenerate positions within the van Devanter primers. DNA polymerase of some herpesviruses were not amplified at all by the dI-substituted primers (e.g. CMV). Using a mixture of dI-substituted and unsubstituted primers, Ehlers found that the mixed primer set improved overall performance for herpesviruses from a range of species. Ehlers demonstrated that 6 of the 8 human herpesviruses (HSV-1, HSV-2, VZV, EBV, CMV and HHV-8) could be amplified by this method, while reducing the virus-related variability in the limit of detection. However, the important issues of assay complexity and turn-around time were not addressed since Ehlers, like van Devanter, intended to utilize the assay primarily to support research rather than clinical analysis.
Colimon et al. (1996) developed the use of “stair primers” to allow PCR amplification of viral genomes with frequent point mutations, such as HIV and hepatitis C virus. Minjolle et al. (1999) adopted the use of these “stair primers” for herpesvirus assay, utilizing mixtures of consensus stair primers to amplify DNA polymerase for the detection of 6 of the 8 human herpesviruses (HHV-1, HHV-2, VZV, EBV, CMV and HHV-6). However, the stair primer method requires 11 sets of synthetic primers. Amplicons were detected by virus-specific probe hybridization with chromogenic detection.
Robert et al. (2002) utilized a commercially available kit based on Minjolle's stair primers in a two stage multiplex PCR assay of 6 herpesviruses (HHV-1, HHV-2, VZV, EBV, CMV, and HHV-6) in tear fluid. Samples were amplified using the Argene Herpes Consensus Generic Kit. Amplicons positive for herpesvirus were typed with the Argene Herpes Identification (Hybridowell) Kit by virus-specific probe hybridization with a chromogenic substrate for detection.
Pozo and Tenorio (1999) developed a two-step consensus primer PCR assay for the 6 lymphotropic human herpesviruses. Six pairs of primers were used in a first PCR step to produce a virus-specific 194 bp amplicon of the DNA polymerase gene. Then six pairs of primers were used in a second PCR step in which the reverse primer targets a highly conserved region of each amplicon, and the forward primer governs a difference in amplicon size (e.g. 54-122 bp). Subsequent gel electrophoresis with ethidium bromide-staining allowed typing of each band by its migration rate on the gel. A limit of detection of 10-100 copies for the 6 lymphotropic herpesviruses was reported.
Johnson et al. (2000) recently developed a two-step PCR-based assay for detection and species identification of human herpesviruses. Two consensus primer pairs were used, one for the three α-herpesviruses, the other pair for the five β and γ-herpesviruses. The primer pairs bracketing a highly conserved region of the DNA polymerase gene allowed amplification of all eight major human herpesviruses at a limit of detection of 10-100 copies, with the exception of CMV that had a limit of detection of 400 copies. Johnson also claimed to be the first to differentially diagnose HHV-6A and HHV-6B variants, although this does not appear to have clinical significance. Johnson used agarose gel electrophoresis with visual identification of fluorescent ethidium bromide stained bands to identify sample amplicons positive for human herpesvirus. Positive amplicon reaction mixtures were then subjected to two separate restriction endonuclease digestions (BamHI and BstUI). The restriction digests were then subjected to agarose gel electrophoresis and the human herpesvirus species was identified based on the restriction fragment patterns (RFLP) on the two gels. However, the use of dual RFLP is overly complex and time-consuming for the clinical laboratory.
Thus, the prior art is deficient in assays capable of detecting and typing all human herpesviruses in a clinical setting. The present invention fulfills this long-standing need and desire in the art.
TABLE 1Human Herpesvirusesα Herpesvirusesβ Herpesvirusesγ HerpesvirusesShort replicationLong replication cycleVery restrictedcycleHost cell enlargementhost rangeHost cell deathRestricted host rangeEstablish latentReplicate inEstablish latentinfection inbroad range ofinfection in many celllymphoid tissuehost tissuestypes (secretory(lymphotropic)Establish latentglands, kidneys,infection inepithelium,sensory nerveendothelium,gangliamonocytes,(neurotropic)lymphocytes)HSV-1CMVEBVHerpes simplex(congenital infection,(EBV-1 and EBV-2type 1retinitis, pneumonitis)variants)(oral herpes)HHV-6Burkitt lymphoma,HSV-2(6A and 6B variants)nasopharyngealHerpes simplex(roseola, kidneycarcinoma, Hodgkintype 2transplant rejection)lymphoma, AIDS(genital herpes)HHV-7lymphoma, PTLD)VZVHHV-8 (KSHV)(chickenpox,Kaposi's sarcoma,herpes zoster orprimary effusionshingles)lymphoma